Most protein detection assays rely on the efficient separation of target bound detector reagents from non-bound reagents. The assay sensitivity is crucially dependent on this separation and the power of the reporter system used. For the separation, a solid phase is usually employed containing an immobilised affinity reagent for the analyte with a secondary affinity reagent for the detection, as in a sandwich ELISA. In the operation of washing off unbound detector reagents the stringency must be carefully balanced. At high stringency of washing the background is lower at the cost of reduced detection signal of analytes and at low stringency the probability of detection is higher, but at the cost of higher background.
Assays that use a solid phase require considerable time and effort for the analysis. The binding of reagents and analytes to the surface is a time consuming process and the washing steps must be precisely and reproducibly controlled between samples to give exact results. Solid phase assays are also more difficult to automate than homogenous assays.
Assays that do not require a solid phase and the associated washing steps have important advantages. Such homogenous assays exist, for example the scintillation proximity assay (1) and assays based on fluorescence resonance energy transfer (2, 3). However these suffer from a low sensitivity due to poor signal to noise ratios.
In WO 97/00446 an ultrasensitive immunoassay and kit is described using two reagents that are crosslinked if they adhere to an analyte. The coincident binding of two analytes give rise to an interaction product, which may be selected so that it can serve as a template in amplification reactions, increasing specificity through the requirement for dual recognition and reducing the risk for non-specific signals. This immunoassay is performed on a solid support.